Identifying Epigenetic, Chromatin, and Transcriptomic landscapes to Improve SCNT Development in an Animal Model

项目总结 体细胞核移植(SCNT)可以创造出基因完全相同的动物 用于农业用途，是生产生物医学用基因工程动物的有力工具 用于治疗的患者特异性胚胎干细胞(SCNT-ESCs)的应用和生成。然而， 长期开发效率低，限制了其广泛应用和实用价值。最基本的 问题是卵子是如何不完全地对体细胞供体核进行重新编程的。该计划的总体目标 目前的提议概括为两个具体目标：1)确定表观遗传、染色质和转录 体外受精(IVF)胚胎的景观，然后阐明SCNT胚胎是如何偏离这一点的 发展范式；以及2)建立和测试纠正重新编程缺陷和 促进植入前和植入后胚胎的SCNT发育。我们创造了高- 精细分期的试管受精和SCNT牛胚胎和利用尖端技术的高质量开放染色质图谱 单细胞开放染色质图谱研究染色质在胚胎内和胚胎间的异质性 重新编程。这些地图允许我们第一次询问，如何特定的区域 正常着床前发育过程中基因组的开放和关闭，以及SCNT胚胎如何偏离 从这个范例中。我们的初步数据清楚地表明，早期卵裂分期的SCNT胚胎并不 适当地建立开放的染色质，并牵涉到EGA的重要贡献者-转录因子(Tf) DUX是SCNT胚胎的一个主要缺陷。我们假设染色质重编程的失败 在SCNT植入前发育中，植入后发育较晚，且靶向 使用TF表达或植入前筛查的干预将提高SCNT的发育成功率。目标 1包括三个分目标：1.1。确定与试管受精不同的性别特异性SCNT开放染色质区域 胚胎，或表现出性别特异性；1.2。鉴定SCNT胚胎中抗性的体细胞记忆 使用DNA甲基化分析进行重新编程；以及1.3.鉴定SCNT之间的基因表达差异 和体外受精囊胚，并评估它们是否偏向ICM或TE系。根据这些数据，我们将 使用生物信息学分析来确定额外的TF/调节器，以进行进一步的机械解剖。在目标2中， 将测试三个分目标：2.1。如果异地强制执行EGA(使用DUX和其他TF)可以重新激活 培养成纤维细胞中的EGA程序；2.2。如果使用转铁蛋白靶向激活SCNT胚胎中的EGA网络 或TF组合(从2.1)将改善重新编程并促进它们的发展和2.3。如果是单身- 早期细胞活检和开放染色质图谱可以预测体外受精和 SCNT胚胎。这些目标将提高农业和生物医学动物的世代效率 为人类临床应用建立模型并建立策略和见解。

PROJECT SUMMARY Somatic cell nuclear transfer (SCNT) allows for the creation of genetically identical animals for agricultural purposes, and is a powerful tool for production of genetically engineered animals for biomedical applications and generation of patient-specific embryonic stem cells (SCNT-ESCs) for therapeutics. However, the low efficiency of full term development limits its wide-spread use and practical utility. The fundamental problem is how the egg incompletely reprograms a somatic donor nucleus. The overall objectives of the current proposal are outlined in the two Specific Aims : 1) Identify epigenetic, chromatin, and transcriptomic landscapes in in vitro fertilized (IVF) embryos and then elucidate how SCNT embryos deviate from this developmental paradigm; and 2) Establish and test new approaches to correct reprogramming defects and improve SCNT development both in preimplantation and postimplantation embryos. We have created high- quality open chromatin maps for carefully-staged IVF and SCNT bovine embryos and leveraged cutting-edge single-cell open chromatin profiling to clarify intra- and inter-embryo heterogeneity in chromatin reprogramming. These maps allow us to interrogate, for the first time, how specific regions of the genome open and close during normal preimplantation development, and how SCNT embryos deviate from this paradigm. Our preliminary data clearly indicate early cleavage staged SCNT embryos do not establish open chromatin properly, and implicate an important contributor to EGA, a transcription factor (TF) DUX, as a major deficiency in SCNT embryos. We hypothesize that failures in chromatin reprogramming during SCNT preimplantation development manifest later in postimplantation development, and that targeted intervention using TF expression or preimplantation screening will improve SCNT developmental success. Aim 1 consists of three subaims: 1.1. Determine sex-specific SCNT open chromatin regions that deviate from IVF embryos, or exhibit sex-specificity; 1.2. Identify somatic memory in SCNT embryos that is resistant to reprogramming using DNA methylation profiling; and 1.3. Identify gene expression differences between SCNT and IVF blastocysts and evaluate if they are biased towards ICM or TE lineages. Based on these data, we will use bioinformatic analyses to identify additional TFs/regulators for further mechanistic dissection. In Aim 2, three subaims will be tested: 2.1. if ectopic enforcement of EGA (using DUX and other TFs) can reactivate the EGA program in cultured fibroblasts; 2.2. If targeted activation of the EGA network in SCNT embryos using TF or TF combinations (from 2.1) would improve reprogramming and facilitate their development and 2.3. If single- cell biopsy and open chromatin profiling at early stages can predict developmental outcomes of both IVF and SCNT embryos. These goals will improve the efficiency of generation of agricultural and biomedical animal models and establish strategies and insights for human clinical applications.